Abstract The overall goal of this grant over the years has been to characterize the detailed mechanism of action of various physiologically important forms of phospholipase A2 (PLA2). During the course of these studies, it has become apparent that the activity of this superfamily of enzymes depends critically on the interaction of the proteins with large lipid aggregates. It appears that the orientation of the enzyme with respect to the plane of the lipid-water interface can have a dramatic effect on activity. The nature of this interaction has been difficult to explore because of the fact that it represents the interaction of two large macromolecules. This has presented challenges for traditional NMR and X-ray crystallographic studies. The activity of many of these enzymes appears to increase when the enzyme is at the lipid-water interface. This activation could also be due to changes in enzyme-lipid orientation or to conformational changes in the enzyme. This renewal application will extend our current studies on the human Ca2+-dependent cytosoloic Group IVA PLA2 (GIVA PLA2) and the human Ca2+- independent cytosoloic Group VIA PLA2 (GVIA PLA2). The GIVA PLA2 has been shown to be the critical enzyme in the initiation of eicosanoid production in many cell types, and it plays an important role in inflammation. The control of this critical enzyme has been shown to be modulated by three agents, i.e. Ca2+, ceramide 1-phosphate, and phosphatidylinositol 4,5-bisphosphate. The overall objective of the current grant proposal is to understand how these agents affect the conformation of the enzyme and the orientation of the enzyme with respect to the lipid-water interface and to acertain how these structural components affect enzyme function. The GVIA PLA2 is responsible for remodeling of membrane phospholipids in cells and plays critical roles in the cellular regulation of various diseases. We will determine exactly how it interacts with membranes. Along with traditional biochemical, molecular biological, and kinetic approaches, we will employ amide hydrogen/deuterium exchange-mass spectrometry (DXMS). It is rapidly becoming clear that this technique can tackle many structural questions about how proteins act in solution that cannot be addressed easily by NMR or X- ray crystallography. We will expand the use of the DXMS technique to explore the interactions of these enzymes with large lipid interfaces. We also will use surface plasmon resonance and our detailed surface dilution kinetic model to study the functional aspects of these questions. The results obtained with the GIVA PLA2 will generate important general information on how soluble enzymes interact with lipid-water interfaces. To aid in understanding these general issues, similar amide hydrogen/deuterium exchange studies will be conducted on the GIA PLA2 and two other proteins that associate with the lipid-water interface but also insert into and even through lipid bilayers.